1. Field of the Invention
The field of this invention is cholesterol regulation.
2. Background
Oxysterols are formed by the hydroxylation of the side chain of cholesterol. This modification renders the sterol more hydrophilic and confers two important biological properties. First, the increased hydrophilicity enhances the ability of the oxysterol to cross membranes and thereby facilitates its movement between intracellular compartments, cells and tissues. Second, oxysterols delivered in ethanol to cultured cells, are potent regulators of the expression of genes involved in sterol and fatty acid metabolism (1,2).
The enhanced solubility of oxysterols is exploited by the body to maintain cholesterol homeostasis. In several tissues and cell types, including the brain, kidney, endothelium, and macrophages, cholesterol is converted into oxysterols that subsequently traverse the plasma membrane and are transported to the liver (3-5). In the liver, they are converted into bile acids by a newly described biosynthetic pathway (6). These bile acids are essential for normal lipid and fat-soluble vitamin metabolism (7).
Oxysterols are both positive and negative regulators of gene expression. As positive effectors, they bind to and activate the nuclear receptor LXR (8), which in turn increases transcription of the cholesterol 7xcex1-hydroxylase gene (9). This activation stimulates the conversion of cholesterol into bile acids (10). Mutation of the LXR gene in mice causes a loss of 7xcex1-hydroxylase gene induction and a buildup of cholesterol in the liver (11). As negative regulators, oxysterols suppress the cleavage of two transcription factors known as sterol regulatory element binding proteins-1 and -2 (SREBP-1 and -2) (12). These proteins are synthesized as inactive precursors in the membrane compartment of the cell. When intracellular cholesterol levels decline, SREBPs are cleaved to release amino-terminal fragments that migrate to the nucleus and activate the transcription of a network of genes involved in cholesterol synthesis and supply (12). This activation in turn restores intracellular cholesterol levels.
Several oxysterols occur naturally, including 25-hydroxycholesterol(cholest-5-ene- 3xcex2,25-diol), 24-hydroxycholesterol(cholest-5-ene-3xcex2,24-diol), and 27-hydroxycholesterol(cholest-5-ene-3xcex2,27-diol) (13). Of these three oxysterols, 25-hydroxycholesterol is the most potent regulator of gene transcription when assayed in vitro (1,2,9,11). Hence, 25-hydroxycholesterol biosynthetic enzymes would provide attractive targets for therapeutic inhibitor development, i.e. novel hypocholesteremic agents: by blocking 25-hydroxycholesterol synthesis, SREBPs remain in their active forms and stimulate expression of the LDL receptor, which in turn extracts LDL from the plasma, lowering serum cholesterol.
The invention provides methods and compositions relating to cholesterol 25-hydroxylase polypeptides having cholesterol 25-hydroxylase-specific structure and activity, related polynucleotides and modulators of cholesterol 25-hydroxylase function and serum cholesterol. For example, the subject cholesterol 25-hydroxylase polypeptides and polynucleotides can be used to regulate cholesterol 25-hydroxylase activity, and hence serum cholesterol in a mammalian host. The polypeptides may be recombinantly produced from transformed host cells from the subject cholesterol 25-hydroxylase polypeptide encoding nucleic acids or purified from natural sources such as mammalian cells. The invention provides isolated cholesterol 25-hydroxylase hybridization probes and primers capable of specifically hybridizing with natural cholesterol 25-hydroxylase genes, cholesterol 25-hydroxylase-specific binding agents such as specific antibodies, agonists and antagonists, cholesterol 25-hydroxylase transcriptional regulators, and methods of making and using the subject compositions in diagnosis (e.g. genetic hybridization screens for cholesterol 25-hydroxylase transcripts), therapy (e.g. cholesterol 25-hydroxylase inhibitors to modulate serum cholesterol) and in the biopharmaceutical industry (e.g. as immunogens, reagents for isolating natural 25-hydroxylase genes and transcripts, reagents for screening chemical libraries for lead pharmacological agents, etc.).
The nucleotide sequence of natural human and mouse genes encoding natural human and mouse cholesterol 25-hydroxylase polypeptides are shown as SEQ ID NOS:1 and 3, their natural transcript cDNAs are SEQ ID NO:1, nucleotides 1-1355 and SEQ ID NO:3, nucleotides 1173-2526, respectively, each with an additional 3xe2x80x2 poly A tail, and their full translates are shown as SEQ ID NOS:2 and 4, respectively.
The cholesterol 25-hydroxylase polypeptides of the invention include fragments of SEQ ID NOS:2 and 4 having human cholesterol 25-hydroxylase-specific amino acid sequence, binding specificity and function. Preferred polypeptides comprise at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO:2, wherein such polypeptides and/or consecutive residues are not contained in any conceptual translate of murine ESTs AA289153 and AA285796, nor human ESTs AIO81548, WO1328, and N45640. The subject domains provide cholesterol 25-hydroxylase domain specific activity or function, such as cholesterol 25-specific hydroxylase or hydroxylase inhibitory activity, SCAP (28) binding or binding inhibitory activity, and/or cholesterol 25-hydroxylase specific antibody binding or binding inhibitory activity.
Cholesterol 25-hydroxylase-specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays, e.g. binding assays. The term binding assay is used generically to encompass any assay, including in vitro, cell-cuture or animal-based assays (e.g. using gene therapy techniques or with transgenics), etc. where the molecular interaction of a cholesterol 25-hydroxylase polypeptide with a specific binding target is evaluated. The binding target may be a natural intracellular binding target such as a cholesterol 25-hydroxylase substrate, a cholesterol 25-hydroxylase regulating protein or other regulator that directly modulates cholesterol 25-hydroxylase activity or its localization; or non-natural binding target such as a specific immune protein such as an antibody, or an cholesterol 25-hydroxylase specific agent such as those identified in screening assays such as described below. Cholesterol 25-hydroxylase-binding specificity may be assayed by hydroxylase activity, hydroxylase activity inhibition (e.g. ability of the subject polypeptides to function as negative effectors in cholesterol 25-hydroxylase-expressing cells), by binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by immunogenicity (e.g. ability to elicit cholesterol 25-hydroxylase specific antibody in a heterologous host such as a mouse, rat, goat or rabbit), etc.
In a particular embodiment, the subject polypeptides provide cholesterol 25-hydroxylase-specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, the subject polypeptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of cholesterol 25-hydroxylase-specific antibodies is assayed by solid phase immunosorbant assays using immobilized cholesterol 25-hydroxylase polypeptides of SEQ ID NOS:2 and 4, see, e.g. Table 1.
The claimed cholesterol 25-hydroxylase polypeptides are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state. Isolated polypeptides encompass cholesterol 25-hydroxylase polypeptides covalently joined to a non-natural or heterologous component, such as a non-natural amino acid or amino acid sequence or a natural amino acid or sequence other than that which the polypeptide is joined to in a natural protein, are preferably in solution, and preferably constitute at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and pure polypeptides constitute at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The polypeptides may be covalently or noncovalently part of a larger complex, such as larger polypeptides and/or various conjugates, etc. The polypeptides may be synthesized, produced by recombinant technology, or purified from mammalian, preferably human cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.
The invention provides binding agents specific to the claimed cholesterol 25-hydroxylase polypeptides, including substrates, agonists, antagonists, natural intracellular binding targets, etc., methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, specific binding agents are useful in a variety of diagnostic and therapeutic applications, especially where disease or disease prognosis is associated with unoptimized utilization of a pathway involving one or more of the subject polypeptides, e.g. cholesterol regulation. Novel cholesterol 25-hydroxylase-specific binding agents include cholesterol 25-hydroxylase-specific receptors, such as somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory) and other natural intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as in vitro, cell-based and animal-based binding assays described herein, or otherwise known to those of skill in the art, etc. Agents of particular interest modulate cholesterol 25-hydroxylase function, e.g. cholesterol 25-hydroxylase-dependent hydroxylation, and including dominant negative deletion mutants, etc. Accordingly, the invention also provides methods for modulating cholesterol regulation in a cell comprising the step of modulating cholesterol 25-hydroxylase activity, e.g. by contacting the cell with a substrate, agonist or antagonist of a resident cholesterol 25-hydroxylase, a dominant negative cholesterol 25-hydroxylase deletion mutant, or cholesterol 25-hydroxylase polynucleotide (below).
The amino acid sequences of the disclosed cholesterol 25-hydroxylase polypeptides are used to back-translate cholesterol 25-hydroxylase polypeptide-encoding polynucleotides optimized for selected expression systems (Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166) or used to generate degenerate primers and probes for use in the isolation of natural cholesterol 25-hydroxylase-encoding nucleic acid sequences (see, e.g. xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.). The terms polynucleotide and nucleic acid are used interchangeably to refer to any polymer of nucleotides, without restriction by length. Cholesterol 25-hydroxylase-encoding polynucleotides may be used in cholesterol 25-hydroxylase-expression vectors and incorporated into recombinant host cells, e.g. for expression and screening, transgenic animals, e.g. for functional studies such as the efficacy of candidate drugs for disease associated with cholesterol 25-hydroxylase-modulated cell function, etc.
The invention also provides polynucleotides, e.g. hybridization probes and replication/amplification primers, comprising a cholesterol 25-hydroxylase cDNA specific sequence comprising at least one of SEQ ID NO:1 or 3 and polynucleotides comprising a nucleotide sequence sufficient to effect specific hybridization to SEQ ID NO:1 or 3 and not contained in any of murine ESTs AA289153 and AA285796, nor human ESTs A1081548, WO1328, and N456401, such specifically hybridizing polynucleotides including polynucleotides comprising one or more fragments of SEQ ID NO:1 or 3. Such polynucleotides and fragments are at least 12, preferably at least 24, more preferably at least 48, more preferably at least 96 and most preferably at least 182 nucleotides in length. Demonstrating specific hybridization generally requires stringent conditions, for example, hybridizing in a buffer comprising 30% formamide in 5xc3x97SSPE (0.18 M NaCl, 0.01 M NaPO4, pH 7.7, 0.001 M EDTA) buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE; preferably hybridizing in a buffer comprising 50% formamide in 5xc3x97SSPE buffer at a temperature of 42xc2x0 C. and remaining bound when subject to washing at 42xc2x0 C. with 0.2xc3x97SSPE buffer at 42xc2x0 C. Specifically hybridizing polynucleotides are readily identified in convenient gel-based assays; for example, polynucleotides comprising SEQ ID NOS:5-10 are shown to specifically hybridize with SEQ ID NO:1 and/or 3 under the foregoing preferred hybridization conditions. Cholesterol 25-hydroxylase nucleic acids can also be distinguished using alignment algorithms, e.g. nucleic acids having a BLASTn 2.0 (See, http://www.ncbi.nlm.nih.gov/BLAST/) bit score of at least 100, preferably at least 200 more preferably at least 400, most preferably at least 800 with a significance of at least e-50, preferably at least e-75, more preferably at least e-100, most preferably at least e-125, using default program settings.
The invention also provides regulators of cholesterol 25-hydroxylase gene expression, including natural upstream (5xe2x80x2) cholesterol 25-hydroxylase gene transcriptional regulatory elements. Native cholesterol 25-hydroxylase promoter elements may be truncated and recombined to generate novel cholesterol 25-hydroxylase transcriptional regulatory elements. In a particular embodiment, the invention provides promoters comprising a cholesterol 25-hydroxylase gene specific sequence comprising SEQ ID NO:3, nucleotides 1-1182 and promoters comprising a nucleotide sequence that effects specific hybridization to SEQ ID NO:3, nucleotides 1-1182, provides one or more cholesterol 25-hydroxylase promoter activities and comprises one or more fragments of SEQ ID NO:3, nucleotides 1-1182. Specifically hybridizing polynucleotides are readily identified in convenient gel-based assays; for example, polynucleotides comprising SEQ ID NOS:11-16 are shown to specifically hybridize with SEQ ID NO:3, nucleotides 1-1182 under the foregoing preferred hybridization conditions. Such polynucleotides and fragments are at least 12, preferably at least 24, more preferably at least 48, more preferably at least 96, and most preferably at least 182 nucleotides in length. Generally, such elements comprise one or more cholesterol 25-hydroxylase promoter DNA binding protein and/or transcription factor binding sites, examples of which are provided in Table 3.
Transcriptional regulatory activity is conveniently assayed in transcriptional reporter assays. For example, Table 4 provides cholesterol 25-hydroxylase gene promoter constructs which can regulate expression of luciferase enzymatic activity in CaPO4 transfected 293 or HeLa cells. For these assays, cells are harvested 18 hrs post transfection and assayed for luciferase.
The subject polynucleotides are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total polynucleotides present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that to which it is joined on a natural chromosome. Recombinant polynucleotides comprising the nucleotide sequence of SEQ ID NO:1 or 3, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, which is at a terminus or is immediately flanked by a sequence other than that to which it is joined on a natural chromosome. While the polynucleotides are usually RNA or DNA, it is often advantageous to use polynucleotides comprising other bases or nucleotide analogs to provide modified stability, etc.
The subject nucleic acids find a wide variety of applications including use as translatable transcripts, hybridization probes, PCR primers, diagnostic nucleic acids, etc.; use in detecting the presence of cholesterol 25-hydroxylase genes and gene transcripts and in detecting or amplifying nucleic acids encoding additional cholesterol 25-hydroxylase homologs and structural analogs. In diagnosis, cholesterol 25-hydroxylase hybridization probes find use in identifying wild-type and mutant cholesterol 25-hydroxylase alleles in clinical and laboratory samples. Mutant alleles are used to generate allele-specific oligonucleotide (ASO) probes for high-throughput clinical diagnoses. In therapy, therapeutic cholesterol 25-hydroxylase nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active cholesterol 25-hydroxylase.
For example, cholesterol 25-hydroxylase nucleic acids are used to modulate cellular expression or intracellular concentration or availability of active cholesterol 25-hydroxylase protein. Cholesterol 25-hydroxylase inhibitory nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed natural cholesterol 25-hydroxylase transcript sequences. Antisense modulation of the expression of a given cholesterol 25-hydroxylase protein may employ antisense nucleic acids operably linked to gene regulatory sequences. Cell are transfected with a vector comprising a cholesterol 25-hydroxylase sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous cholesterol 25-hydroxylase encoding mRNA. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding cholesterol 25-hydroxylase protein may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in expression of the targeted protein. An enhancement in cholesterol 25-hydroxylase expression is effected by introducing into the targeted cell type cholesterol 25-hydroxylase nucleic acids that increase the functional expression of the corresponding gene products. Such nucleic acids may be cholesterol 25-hydroxylase expression vectors, vectors that upregulate the functional expression of an endogenous allele, or replacement vectors for targeted modification of endogenous mutant or wild type alleles. Techniques for introducing the nucleic acids into viable cells are known in the art and include retroviral-based transfection, viral coat protein-liposome mediated transfection, etc.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents active at the level of a cholesterol 25-hydroxylase modulatable cellular function and/or cholesterol 25-hydroxylase gene expression, including transcription. A wide variety of assays for transcriptional modulators or binding agents are provided including labeled in vitro ligand binding or hydroxylation assays, immunoassays, cell-based reporter assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for animal and human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
A wide variety of assays for cholesterol 25-hydroxylase transcriptional regulators are provided including cell-based transcription reporter assays, gel-based or solid-phase promoter-protein binding assays, etc. In a particular embodiment, cholesterol 25-hydroxylase promoter-luciferase reporter constructs are used to transfect cells such as HeLa cells for cell-based transcription assays. Specifically, HeLa cells are plated onto microtiter plates and used to screen libraries of candidate agents for lead compounds that modulate the transcriptional regulation of the cholesterol 25-hydroxylase gene promoter, as monitored by luciferase expression.
A wide variety of assays for binding agents, i.e. screens for compounds that modulate cholesterol 25-hydroxylase interaction with a natural cholesterol 25-hydroxylase binding target are also provides. These assays employ a mixture of components including a cholesterol 25-hydroxylase polypeptide, which may be part of a fusion product with another polypeptide, e.g. a peptide tag for detection or anchoring, etc. The assay mixtures comprise a natural intracellular cholesterol 25-hydroxylase binding target. In a particular embodiment, the binding target is a cholesterol 25-hydroxylase substrate, agonist, antagonist or regulator. In the case of polypeptide regulators, one may use portions (e.g. peptides) thereof so long as the portion provides binding affinity and avidity to the subject cholesterol 25-hydroxylase polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like, salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used. In a preferred embodiment, the mixture is provided as a cell line expressing the cholesterol 25-hydroxylase polypeptide in a regulated fashion, as the TR3202a cells described below, or in a cell extract, wherein cholesterol 25-hydroxylase expression is induced and radiolabeled cholesterol substrate is added to the cells.
The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the cholesterol 25-hydroxylase polypeptide specifically binds the cellular binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings, and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening. In a preferred embodiment, the binding effects the conversion of the radiolabeled substrate to 25-hydroxycholesterol.
After incubation, the agent-biased binding between the cholesterol 25-hydroxylase polypeptide and one or more binding targets is detected by any convenient way. A variety of methods may be used to detect the change depending on the nature of the product and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. For the preferred cell- or extract-based cholesterol 25-hydroxylase assays, xe2x80x98bindingxe2x80x99 is generally detected by a change in the hydroxylation of a cholesterol 25-hydroxylase substrate, such as the conversion of radiolabeled cholesterol to 25 hydroxycholesterol, e.g. by thin layer chromatography.
A difference in the binding affinity of the cholesterol 25-hydroxylase to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the cholesterol 25-hydroxylase to the cholesterol 25-hydroxylase binding target. Analogously, in the cell-based assay also described below, a difference in cholesterol 25-hydroxylase-dependent transcriptional activation in the presence and absence of an agent indicates the agent modulates cholesterol 25-hydroxylase function. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The following experimental section and examples are offered by way of illustration and not by way of limitation.